The main objectives of this VA Merit Review renewal proposal are to gain a better understanding of the molecular pathogenesis of bladder cancer and to utilize this information to devise improved diagnostic, preventive and therapeutic approaches for this disease. Bladder cancer is the fifth most prevalent cancer among the Americans and the costliest cancer to treat on a per case basis. However, despite its prevalence, clinical importance and high medical expenses, bladder cancer has historically lagged behind many other cancer types in research endeavors and hence new discoveries. As a result, highly reliable diagnostic and prognostic biomarkers and effective treatment modalities remain scarce. Research supported by this Merit Review Award has been focused on the molecular and genetic events that are capable of driving bladder cancer formation and progression along the divergent pathways. The approaches have concentrated on the development and analyses of genetically engineered mice by expressing or abrogating genes of interest or both in a bladder-specific and time-controlled manner. These studies have been highly informative regarding the tumorigenic ability or the lack thereof of candidate genes in bladder cancer formation, the gene-pathway association, the sequence of tumor development in the bladder, the collaborative relationship of different oncogenic events, the unique context of bladder epithelium in tumorigenesis and the mechanisms of tumor suppressor gene inactivation. Results from these studies underscore the need of combining different biomarkers for more accurate diagnosis and prediction of prognosis. These studies have also been instrumental in identifying potential targets that can be further explored for therapeutic intervention for bladder cancer clinically. This renewal proposal builds on the broad foundation established during the past funding period to tackle several new, hypothesis-driven and highly focused questions. Specifically, the renewal proposal will investigate, in three Specific Aims, the role of pyruvate kinase isoform 2 (PKM2), found during the preliminary studies to be highly expressed in mouse bladder cancer models, human bladder cancer cell lines and specimens, in bladder tumor initiation and progression. Specific Aim 1 is designed to identify signals in the mitogenic pathways that are capable of up-regulating and activating the growth-promoting functions of PKM2, an extremely important but under-explored area. Both specific-target-based and high throughput siRNA knock- down approaches will be employed with cultured human bladder cancer cell lines. Specific Aim 2 will investigate the in vivo role of PKM2 in bladder tumor initiation using complementary over-expression as well as knock-down approaches. Novel transgenic mouse lines will be engineered to over-express PKM2 and then be crossed to existing transgenic lines that bear tumor-precursor lesions, thus providing opportunity to determine whether PKM2 promotes bladder tumor formation. Additionally, PKM2 will be knocked down using isoform- specific shRNA and its effects on tumor growth will be examined in vitro and in vivo in xenograft and orthotopic models. Specific Aim 3 will evaluate whether inhibition of PKM2 with naturally occurring products, alone or in combination with chemotherapeutics, helps slow tumor growth in low-grade non-invasive as well as high-grade invasive bladder tumor variants. Together, these three series of studies should greatly enhance our understanding of the role of PKM2 over-expression in bladder tumorigenesis and progression and the prospect of using PKM2 as a diagnostic biomarker and/or a therapeutic target for bladder cancer treatment.